Cellular distribution and inducible degradation and resynthesis of IκB-α in human neutrophils. (A) Unstimulated neutrophils or monocyte-enriched mononuclear cell suspensions from the same donor were submitted to NP40 lysis, and the resulting nuclear-containing (“N”) and nonnuclear (“NN”) fractions were processed for electrophoresis (2 × 10⁶ cell-equivalents per well) and immunoblotting using an anti-IκB–α antibody (no. 9). For comparative purposes, the immunoblot shown in this panel was performed using samples from the same experiment as the one depicted in Fig 1. This experiment is representative of three. (B) Nonnuclear fractions from resting neutrophils were immunoprecipitated with antisera raised against individual NF-κB/Rel proteins, and the resulting immunoprecipitates (2.5 × 10⁶ cell-equivalents) were processed for immunoblot analysis using an anti-IκB–α antibody (sc-371). For comparative purposes, the immunoblot shown in this panel was performed using samples from the same experiment as the one depicted in Fig 2. This experiment is representative of two. (C) Neutrophils (5 × 10⁶/mL) were cultured at 37°C in the presence of 1 μg/mL LPS or 10 nmol/L fMLP. Aliquots were taken at the indicated times, submitted to nonionic detergent lysis, and the resulting nonnuclear fractions were processed for immunoblot analysis using an anti-IκB–α antibody (sc 371). Each of the experiments depicted in this panel is representative of at least four. pmn, polymorphonuclear neutrophils; mono, autologous monocyte-enriched suspensions; MW, molecular weight markers (in kD); ipp, antiserum used for immunoprecipitation.